Anthrax is a well-known infectious disease caused by a Gram-positive bacterium, Bacillus anthracis. There are three types of anthrax infections: cutaneous, gastrointestinal and inhalation. Inhalation anthrax generally occurs after an incubation time of 1-6 days. After the incubation period, a non-specific flu-like illness ensues for 1-3 days followed by a brief intervening period of improvement. Unfortunately, rapid deterioration follows and death is universal in untreated cases.
Airborne anthrax has long been concerned a major bioterror threat and it has recently been shown that anthrax can be aerosolized and transmitted by mail.
The causative agent of anthrax, Bacillus anthracis, expresses three major extracellular toxin protein components, encoded on its large pXO1 plasmid (Okinaka et al., 1999). Protective antigen (PA) combines with either lethal factor (LF) or edema factor (EF) to form a functional binary toxin (reviewed in Abrami et al., 2005). PA in combination with LF causes death in experimental animals (Smith and Keppie, 1954, Nature 173: 869-870) while PA in combination with EF causes edema in the skin of experimental animals (Stanley and Smith, 1961, J Gen Microbiol 26: 49-66). While none of the proteins is individually toxic, PA combines with either LF or EF to form one of two binary toxins. PA binds to one of two cellular receptors, TEM8 (Bradley et al., 2001; Liu and Leppla, 2003) or CMG2 (Scobie et al., 2003). Upon receptor binding, the 83 kDa PA (PA83) is cleaved at a specific sequence by furin or a furin-like protease, releasing a 20 kDa N-terminal fragment (PA20) while leaving a 63 kDa C-terminal fragment (PA63) bound to the receptor (Singh et al., 1989; Molloy et al., 1992). An LF binding site on PA63 is simultaneously exposed via this cleavage event (Novak et al., 1992). Spontaneous heptamerization of the PA:receptor complex occurs (Milne et al., 1994), allowing competitive, high affinity binding by EF or LF (Cunningham et al., 2002; Mogridge et al., 2002), followed by internalization of the toxin:receptor complex via clathrin-mediated endocytosis (Abrami et al., 2003). Acidification of the endosome produces structural rearrangements in the PA prepore heptamer, leading to pore formation and membrane insertion (Lacy et al., 2004; Santelli et al., 2004), and subsequent release of LF and/or EF into the cytosol (reviewed in Abrami et al., 2005).
PA is the primary antigenic determinant in currently licensed human anthrax vaccines (Turnbull et al., 1986; Leppla et al., 2002; Baillie et al., 2004; Adams et al., 2005). Several recent model studies demonstrate that a strong humoral response to PA contributes to a protective immune response to anthrax (Miller et al., 1998; Pitt et al., 2001; Reuveny et al., 2001; Little et al., 2004), and several regions that serve as targets for neutralizing monoclonal antibodies have been identified (Little et al., 1996; Brossier et al., 2004). The mature 735 amino acid PA protein (GenBank accession number AAT98414) contains four distinct functional domains (Petosa et al., 1997). Domain 1 (residues 1-258) functions in oligomerization of PA and binding to LF and EF (Chauhan & Bhatnagar, 2002; Cunningham et al., 2002; Lacy et al., 2004), and contains the sequence 164RKKR167, which serves as the furin cleavage site (Singh et al., 1989; Molloy et al., 1992). Domain 2 (residues 259-487) functions in pore formation, heptamerization, membrane insertion, and translocation of EF and LF (Benson et al., 1998; Miller et al., 1999; Singh et al., 1994). Domain 3 (residues 488-595) functions in oligomerization (Mogridge et al., 2001), while domain 4 (residue 596-735) functions in binding the cellular receptor (Singh et al., 1991; Varughese et al., 1999; Santelli et al., 2004).
Multiple MAbs that target different regions of PA and neutralize LeTx in vitro have been previously characterized. Several MAbs target epitopes in domain 4, and neutralize the toxin by preventing PA from binding to its cellular receptor (Little et al., 1988; Little et al., 1996; Brossier et al., 2004). Other MAbs target epitopes in regions spanning the interfaces between domains 1 and 2 and domains 3 and 4, and prevent LF from interacting with PA at the cell surface (Little et al., 1996), or target epitopes in domain 2, preventing cleavage of PA83 to PA63 (Brossier et al., 2004). To identify unique neutralizing epitopes in PA, MAbs were raised in mice using whole rPA as the immunogen, and their affinities and epitope specificities were characterized.
PCT application WO 02/100340 teaches a vaccine comprising recombinant PA which may be combined with LFn, a Lethal Factor deletion which has the C-terminal 47 amino acids removed, thereby eliminating the lethal toxin forming activity.
Published patent application US 2004/0028695 teaches an expression vector for a “27 kDa N-terminal PA deletion mutant PA27. This mutant contains amino acid 498-735 of PA and the purpose of this mutant is to create a smallest PA deletion mutant to be used as an effective antigen.” In one embodiment of the invention, a fusion protein comprising the N-terminal domain 1 of LF and the C-terminal domains 3 and 4 of PA are fused, with domain 3 of PA acting as “a spacer region . . . to keep the correct folding structures of the other two domains from LF and PA”.
These vaccines are based on the observation that the protective efficacy of PA is greatly increased if small quantities of LF or EF are incorporated into the vaccine (Pezard et al., 1995, Infect. Immun. 63: 1369-1372). However, it is believed that this also happens to be the primary cause of toxigenicity and reactogenicity of the vaccines.
Published patent application US 2004/0009945 teaches an anthrax vaccine wherein the PA coding sequence is inserted into a VEE virus vector in place of VEE virus structural genes.
PCT application WO 03/048390 teaches an anthrax vaccine which comprises PA, LF and EF together wherein these proteins have been rendered non-toxic by introducing mutations which affect the biological activity of the proteins without affecting their structure or immunogenicity.
Clearly, there remains a need for an anthrax vaccine which has a well defined composition and has minimal, if any, side effects.